Many pathological processes such as thrombosis and hemorrhages, systemic inflammation and cytokine shock, pathologies of transport of lipids and other biological compounds, are initiated or/and develop in the blood. Accordingly, activity of therapeutic agents for optimal interventions in these processes must be localized in this compartment. However, most drugs do not circulate for a sufficient time in the bloodstream because of their uptake by liver, renal filtration and diffusion into the tissues via a vascular wall that is permeable for many therapeutic agents via active and passive transport across endothelium and peri-cellular permeability of endothelial monolayer. Current means for retention of drugs in the bloodstream, such as PEG-ylation or encapsulation into PEG-ylated drug carriers (e.g., liposomes) improve their blood solubility and inhibit recognition by the immune system, thus prolonging their longevity in circulation. However, all previous means for drug retention in bloodstream, including PEG-technology, employed relatively small compounds and carriers (e.g., liposomes of ˜100 nm diameter) that can diffuse in tissues (leading to side effects and drug elimination) and uptake by clearing organs (liver and kidney). Therefore, despite relatively easy access to many pathological targets in blood, repeated administrations of high doses of a drug are needed to compensate for clearance. This complicates therapies and leads to adverse effects. The general problem of inadequate therapeutic interventions in the pathological processes in blood has not been resolved.
For example, hemostasis, the sealing of damaged blood vessels by mural clots, prevents bleeding. Thrombosis, pathological intravascular occlusion by clots, can cause tissue ischemia and damage leading to acute myocardial infarction (AMI), ischemic stroke, pulmonary embolism and ischemic peripheral vascular disease, among other conditions. Thrombosis is the leading cause of mortality and disability in the United States. Thrombi are prone to recur within hours to days after an AMI or stroke and the risk is great after transient ischemic attack or pulmonary embolism and in immobilized patients. Thrombosis is also a common and dangerous complication of surgery that is especially difficult to manage due to the risk of acute bleeding at the operative site. Invasive interventions (e.g., angioplasty and carotid endarterectomy) may be complicated by formation of small clots that embolize to the brain and cause neurological dysfunction.
Therefore, situations in which patients are at highest risk for occurrence or recurrence of thrombosis, and means to identify such high-risk patients are known. Nevertheless, safety and efficacy of current prevention and management of thrombosis attained with current anti-thrombotic agents (ATAs) and means for their delivery remain inadequate. Anti-platelet and anticoagulant agents provide only limited prophylaxis and pose considerable risk of bleeding. Emergency therapy of thrombosis employs vascular injection of plasminogen activators (PAs), proteases (MW 30-60 kD) that generate plasmin, which cleaves fibrin clots and thus restores perfusion. However, inadequate circulation time (blood clearance within <15 min), inactivation by plasma inhibitors such as PAI-1 and impermeability of occlusive clots restrict the effectiveness of therapeutic fibrinolysis by PAs. Significant pharmacological doses of a PA (e.g., ˜100 mg of tissue type plasminogen activator, tPA) are needed to overcome its inefficiency and achieve fibrinolysis locally. As a result, excess drug diffuses into hemostatic mural clots within minutes after infusion, causing bleeding into tissues. Bleeding into the CNS may cause cerebral hemorrhage. In addition, tPA diffusing into the CNS causes neuronal toxicity and inflammation in the brain. Due to the risk of bleeding and collateral CNS damage, fibrinolytics and anti-coagulants are not used in the post-operative period and in over 95% of stroke patients.
In addition, the only currently employed use of PA, i.e., post-thrombosis, is marred by inevitable delays (time needed for diagnosis, transportation, injection and clot dissolution, slowed by clot impermeability). This delay increases the risk of ischemia-reperfusion (I/R) injury that worsens outcome. Unfortunately, available prophylactic drugs (e.g., anticoagulants and thrombin inhibitors) do not provide adequate protection against thrombosis. First, they are not completely effective due to redundancy of thrombotic mechanisms (e.g., anticoagulants do not prevent platelet activation and anti-platelet agents do not inhibit coagulation). Second, many prophylactic agents altering metabolism of pro-coagulant factors (e.g., inhibitors of the synthesis of vitamin-K dependent coagulation factors, warfarin) require a substantial time to develop an effect (e.g., approximately 36 hours after warfarin administration). Such time frames are not suitable for thromboprophylaxis in acute settings. Third, all of these drugs predispose to bleeding (in addition to other side effects), resulting in a high danger of bleeding, which limits drug utility and dosing especially in the acute settings.
PAs currently in use cannot be used for prophylaxis because of their unfavorable pharmacokinetics and therefore the need to administer potentially dangerous doses of drug for a prolonged period of time. At clinically relevant doses, these PAs cleave fibrin in both hemostatic as well as newly formed clots, which predisposes to bleeding, whereas their penetration into the surrounding tissues causes toxic effects including collateral damage in the central nervous system (CNS).
Targeting of ATAs to clot components (e.g., by chemical conjugation or recombinant fusion with antibodies or antibody fragments that bind to fibrin or activated platelets) does not obviate the failure of ATAs to distinguish between hemostatic and pathological thrombi. In addition, these ATAs with high affinity to clots less effectively permeate into the clots due to enhanced retention on the clot surface, which impedes thrombolysis.
Other research has involved the targeting of plasminogen activators to the vascular lumen (endothelial targeting) using a PECAM-1 single-chain scFv (Ding, et al., Blood 2005, 106(13):4191-4198. See also Ding, et al., Molecular Interventions April 2006, 6(2): 98-112). Targeting of ATAs, such as PAs, to the surface of endothelium by means of fusion proteins consisting of anti-cell adhesion molecule (anti-CAM) scFvs and ATAs may be employed for a prophylactic administration of PAs, which would preferentially dissolve newly formed pathological clots in the vascular area of interest, hence reduce danger of hemorrhage caused by dissolution of existing hemostatic clots. This approach seems ideal for prophylaxis of local thromboses after ischemia or reperfusion, such as in organ transplantation. Nevertheless, this approach is also not ideal for thromboprophylaxis of many other pro-thrombotic states (AMI, TIA, PE). Targeting of CAMs on endothelium is relevant only to highly vascularized organs such as lungs. Also, targeting to CAMs cannot fully prevent endocytosis or transcytosis of ATA from luminal to adventitial side of the vessels which will decrease the duration of the prophylactic effect of the drug.
By targeting ATAs, such as PAs, to red blood cells (RBCs), which can be attained by chemical conjugation of ATAs with antibodies that bind to RBCs, the half-life of the drug in circulation can be prolonged from minutes to days. ATA/RBC complexes selectively lyse newly formed clots because they are incorporated during clot development, and their size prevents penetration of pre-existing clots. This drastically diminishes the risk of bleeding. In addition, coupling to a RBC prevents drug permeation into tissues including the CNS.
However, this approach for selective lysis of newly formed clots has several disadvantages for clinical application. For example, current anti-CR1 conjugates (e.g., anti-CR1/tPA, targeted to a specific RBC determinant, CR-1), such as those described in Zaitsev, et al. (Blood, 108(6):1895-1902 (May 30, 2006)) are not preferred therapeutically for a number a reasons, in addition to those described in the preceding paragraph. Furthermore, the CR1 expression level in humans varies from 300 to 1,500 copies per RBC and ˜15% of humans are CR1 negative. Therefore, targeting CR1 may provide insufficient dosing in some cases and will not be useful in CR1-negative patients. Targeting of a highly expressed determinant on RBC with bivalent antibodies can cause RBC aggregation. Therefore, this approach maybe useful only for targeting ATAs to low-abundance RBC determinants, which limits dosing that may be insufficient in the cases of excessive thromboses. Also, Fc fragment of antibodies can activate complement, promote clearance or signal through Fc receptors or induce an immune response in the host. The synthetic chemistry limits yield and homogeneity of the ATA/antibody conjugates, thereby restricting their clinical utility.
In addition, thrombosis is closely intertwined with vascular inflammation. In many cases one of these conditions leads to another and both mutually propagate each other, further aggravating the outcome.
There exists a need for safe and clinically applicable compositions for effective treatment of thrombosis and associated pathological conditions including vascular inflammation.